Cryopreservation

Abstract

Methods and materials for the cryopreservation of cellularised scaffolds used for therapeutic or pharmacological testing purposes that provide a cultured scaffold on which cells have been seeded, equilibrate the cellularised scaffold with a cryopreservative composition comprising culture medium and between 5 and 30% of a cryoprotectant such as DMSO, freeze the equilibrated cellularised scaffold by reducing the temperature continuously by about −1° C./minute to about −80° C., and store the frozen cellularised scaffold at a temperature of between −135° C. and −198° C.

1 . A method for the cryopreservation of a cellularised scaffold, which method comprises:
 (i) providing a cellularised scaffold;   (ii) equilibrating said cellularised scaffold with a cryopreservative composition comprising culture medium and between 5 and 30% of a cryoprotectant;   (iii) freezing the equilibrated cellularised scaffold by reducing the temperature at between −0.8° C. and −1.2° C./minute to between −78° C. to −82° C.;   iv) storing the frozen cellularised scaffold at a temperature of between −135° C. and −198° C.   
     
     
         2 . A method as claimed in  claim 1  wherein step (iii) comprises freezing the equilibrated cellularised scaffold by continuously reducing the temperature at about −1° C./minute to about −80° C. 
     
     
         3 . A method as claimed in  claim 2  wherein step (i) comprises:
 (ia) providing an acellular scaffold; 
 (ib) seeding the acellular scaffold with the cells; 
 (ic) culturing the seeded scaffold to produce said cellularised scaffold. 
 
     
     
         4 . A method as claimed in  claim 3  wherein step (ia) comprises:
 (ia-1) providing a tissue or organ or sample thereof; 
 (ia-2) decellularizing said tissue or organ or sample thereof using one or both of detergents and enzymes to provide an acellular scaffold. 
 
     
     
         5 . A method as claimed in  claim 1  wherein the scaffold is a sheet scaffold, which is preferably tubular. 
     
     
         6 . A method as claimed in  claim 1  wherein the scaffold is derived from an organ or tissue which has been decellularized, wherein the organ is preferably luminal. 
     
     
         7 . A method as claimed in  claim 1  wherein the scaffold is of non-human origin. 
     
     
         8 . A method as claimed in  claim 1  wherein the cellularised scaffold is a tissue engineered oesophagus construct, which is optionally suitable for a neonate or infant. 
     
     
         9 . A method as claimed in  claim 7  wherein the cellularised scaffold is derived from decellularised oesophagus seeded with mesoangioblasts and fibroblasts. 
     
     
         10 . A method as claimed in  claim 9  wherein the culture medium is FBS plus Megacell medium comprising 1% Penicillin Streptomycin; 1% L-Glutamine; 1% non-essential amino acids; 0.1 mM Beta Mercapto Ethanol and 5 ng/ml Basic FGF. 
     
     
         11 . A method as claimed in  claim 1  wherein the scaffold is spherical, cuboid, cylindrical, hexagonal prismatic, conical, frustoconical, or pyramidal. 
     
     
         12 . A method as claimed in  claim 11  wherein the scaffold is cuboid. 
     
     
         13 . A method as claimed in  claim 1  wherein the scaffold is derived from a solid organ which has been decellularized. 
     
     
         14 . A method as claimed in  claim 11  wherein the cellularised scaffold is tissue engineered liver. 
     
     
         15 . A method as claimed in  claim 14  wherein the cellularised scaffold is a decellularised liver tissue seeded with human hepatic cells, which are optionally HepG2 cells. 
     
     
         16 . A method as claimed in  claim 1  wherein the scaffold is a hydrogel scaffold. 
     
     
         17 . A method as claimed in  claim 16  wherein the cellularised scaffold is a hydrogel scaffold seeded with human hepatic cells, which are optionally HepG2 cells. 
     
     
         18 . A method as claimed in  claim 17  wherein the culture medium comprises FBS plus a liver-cell supporting medium. 
     
     
         19 . A method as claimed in  claim 14  wherein the cellularised scaffold is liver model tissue for pharmacological research. 
     
     
         20 . A method as claimed in  claim 1  wherein the cellularised scaffold is tissue engineered lung, intestine, pancreas, muscle or bladder. 
     
     
         21 . A method as claimed in  claim 1  wherein the cryopreservative composition comprises 80% or more culture medium. 
     
     
         22 . A method as claimed in  claim 21  wherein the cryopreservative composition comprises between 5% to 15%, more preferably 8 to 12%, more preferably about 10% cryoprotectant. 
     
     
         23 . A method as claimed in  claim 1  wherein the cryoprotectant is selected from the list consisting of: dimethyl sulfoxide (DMSO); Ethylene glycol; Glycerol; 2-Methyl-2,4-pentanediol; Propylene glycol; Sucrose; Trehalose. 
     
     
         24 . A method as claimed in  claim 1  wherein step (ii) is carried out below ambient temperature, optionally at about 0 to 4° C. 
     
     
         25 . A method as claimed in  claim 1  wherein step (iv) is carried out by placing the equilibrated cellularised scaffold within one or more containers in the vapour phase of liquid nitrogen such as to achieve a temperature of about −160° C. 
     
     
         26 . A method as claimed in  claim 1  wherein step (iv) is carried out for at least 1, 2, 4, or 4 weeks. 
     
     
         27 . A method as claimed in  claim 1  further comprising:
 (v) thawing the frozen cellularised scaffold rapidly in a water bath, optionally at 37° C. 
 
     
     
         28 . A cryopreserved cellularised scaffold obtained according to the method of  claim 1 . 
     
     
         29 . A thawed cryopreserved cellularised scaffold obtained according to the method of  claim 27 . 
     
     
         30 . A kit comprising a cryopreserved cellularised scaffold of  claim 28  and one or more containers. 
     
     
         31 . A kit according to  claim 30 , wherein said kit further comprises instructions or labeling for the use of said kit for therapeutic purposes or for pharmacological research purposes. 
     
     
         32 . A method of treatment of a subject with a chronic illness leading to organ failure, which method comprises:
 (i) providing a cellularised scaffold which is a tissue engineered organ replacement using autologous cells of the subject;   (ii) cryopreserving the organ replacement according to the method of  claim 1 ;   (iii) thawing the organ replacement when it is required by said subject;   (iv) treating said subject using said organ replacement.   
     
     
         33 . A system for providing allogeneic tissue engineered organ replacements, which system comprises:
 (i) providing a plurality cellularised scaffolds which are allogeneic tissue engineered organ replacements using universal donor iPS cells;   (ii) cryopreserving the organ replacements according to the method of  claim 1 ;   (iii) identifying a subject in need to an organ replacement;   (iv) identifying a compatible cryopreserved allogeneic tissue engineered organ replacement;   (v) thawing the organ replacement when it is required by said subject;   (vi) treating said subject using said organ replacement.   
     
     
         34 . A method for providing a three dimensional engineered micro-scaffold for use in pharmacological research purposes, which method comprises:
 (i) providing a cellularised scaffold which is a three dimensional engineered micro-scaffold suitable for pharmacological research purposes;   (ii) cryopreserving the engineered micro-scaffold according to the method of  claim 1 ;   (iii) thawing the micro-scaffold when it is required.   
     
     
         35 . A cellularised scaffold or cryopreserved cellularised scaffold for use in the method or system of  claim 32 . 
     
     
         36 . Use of a cellularised scaffold or cryopreserved cellularised scaffold for the preparation of a medical implant or material, for use in a method or system of  claim 32 .